UNIVERSAL CALIBRATION OF SIZE-EXCLUSION CHROMATOGRAPHY FOR PROTEINS IN GUANIDINIUM HYDROCHLORIDE INCLUDING THE HIGH-MOLECULAR-MASS PROTEINS TITIN AND NEBULIN

The chromatography of polypeptides in 6 M guanidinium hydrochloride (GuHCl) was studied with respect to the measurement of two ultra-large polypeptides, titin and nebulin. These proteins are integral constituents of the muscle structure, responsible for elasticity, and play an important role in muscle function. Both are outside the previously available range of calibration standards. This problem was circumvented by universal calibration in two different solvents, namely denaturing GuHCl conditions for the unknown polypeptides and buffered solutions of viruses under native assembly conditions. The accuracy of the approach was established. Two matrices were tested for their stability towards this solvent change without changing their calibration graphs. For Superose-6 the calibration changed by 10%. TSK-6000PW exhibited a congruent calibration graph for native and denaturing conditions. By extrapolation it was possible to estimate the chain molecular masses for nebulin to be 560 000 and that for T-II, the extractable form of titin, to be 2 000 000.